Complete Genome Sequence of Annamia dubia, filamentous colony-making Chroococcales with the analysis of FraC gene influencing filament integrity.

The complete genome of Annamia dubia was sequenced. The genome size is 4.02 Mbp, including 3886286 bp circular chromosome and four circular plasmids (31516, 42453, 38085 and 24903 bp). It included 3718 protein-coding sequences, 45 tRNA genes, three sets of rRNA genes, a microcystin biosynthesis gene cluster and six CRISPR (clustered regularly interspaced short palindromic repeat). Annamia is the only one genus in the Chroococcales that makes filamentous colonies. FraC and FraG were identified in the genome. These genes are required for the integrity of cell junctions and influencing filament integrity and are thought to be related to colony formation. These genes are first reported from Chroococcales, and may play a significant role in the colony formation of this species. In the phylogenetic tree of the FraC gene, A. dubia was located in the basal position of Oscillatoriales. The GC ratio of FraC gene of A. dubia is very low from the genome and the FraC gene of Microcoleaceae. The presence of these genes in the basal region and the low GC ratio suggests that the FraC gene in this species was introduced by horizontal gene transfer. Since the filamentous colony is a fundamental and important taxonomic feature for the classification of cyanobacteria, the possibility of horizontal transmission of genes involved in filamentous cyanobacterial colonies is an important discovery for the classification of cyanobacteria.

Reorganization of the ancestral sex-determining regions during the evolution of trioecy in Pleodorina starrii.

The coexistence of three sexual phenotypes (male, female and bisexual) in a single species, 'trioecy', is rarely found in diploid organisms such as flowering plants and invertebrates. However, trioecy in haploid organisms has only recently been reported in a green algal species, Pleodorina starrii. Here, we generated whole-genome data of the three sex phenotypes of P. starrii to reveal a reorganization of the ancestral sex-determining regions (SDRs) in the sex chromosomes: the male and bisexual phenotypes had the same "male SDR" with paralogous gene expansions of the male-determining gene MID, whereas the female phenotype had a "female SDR" with transposition of the female-specific gene FUS1 to autosomal regions. Although the male and bisexual sex phenotypes had the identical male SDR and harbored autosomal FUS1, MID and FUS1 expression during sexual reproduction differed between them. Thus, the coexistence of three sex phenotypes in P. starrii is possible.

Characterization of a bloom-associated alphaproteobacterial lineage, 'Candidatus Phycosocius': insights into freshwater algal-bacterial interactions.

Marine bacterial lineages associated with algal blooms, such as the Roseobacter clade, have been well characterized in ecological and genomic contexts, yet such lineages have rarely been explored in freshwater blooms. This study performed phenotypic and genomic analyses of an alphaproteobacterial lineage 'Candidatus Phycosocius' (denoted the CaP clade), one of the few lineages ubiquitously associated with freshwater algal blooms, and described a novel species: 'Ca. Phycosocius spiralis.' Phylogenomic analyses indicated that the CaP clade is a deeply branching lineage in the Caulobacterales. Pangenome analyses revealed characteristic features of the CaP clade: aerobic anoxygenic photosynthesis and essential vitamin B auxotrophy. Genome size varies widely among members of the CaP clade (2.5-3.7 Mb), likely a result of independent genome reductions at each lineage. This includes a loss of tight adherence pilus genes (tad) in 'Ca. P. spiralis' that may reflect its adoption of a unique spiral cell shape and corkscrew-like burrowing activity at the algal surface. Notably, quorum sensing (QS) proteins showed incongruent phylogenies, suggesting that horizontal transfers of QS genes and QS-involved interactions with specific algal partners might drive CaP clade diversification. This study elucidates the ecophysiology and evolution of proteobacteria associated with freshwater algal blooms.

Genomic analysis of an ultrasmall freshwater green alga, Medakamo hakoo.

Ultrasmall algae have attracted the attention of biologists investigating the basic mechanisms underlying living systems. Their potential as effective organisms for producing useful substances is also of interest in bioindustry. Although genomic information is indispensable for elucidating metabolism and promoting molecular breeding, many ultrasmall algae remain genetically uncharacterized. Here, we present the nuclear genome sequence of an ultrasmall green alga of freshwater habitats, Medakamo hakoo. Evolutionary analyses suggest that this species belongs to a new genus within the class Trebouxiophyceae. Sequencing analyses revealed that its genome, comprising 15.8 Mbp and 7629 genes, is among the smallest known genomes in the Viridiplantae. Its genome has relatively few genes associated with genetic information processing, basal transcription factors, and RNA transport. Comparative analyses revealed that 1263 orthogroups were shared among 15 ultrasmall algae from distinct phylogenetic lineages. The shared gene sets will enable identification of genes essential for algal metabolism and cellular functions.

Cryopreservation of two species of the multicellular volvocine green algal genus Astrephomene.

BACKGROUND: Astrephomene is an interesting green algal genus that, together with Volvox, shows convergent evolution of spheroidal multicellular bodies with somatic cells of the colonial or multicellular volvocine lineage. A recent whole-genome analysis of A. gubernaculifera resolved the molecular-genetic basis of such convergent evolution, and two species of Astrephomene were described. However, maintenance of culture strains of Astrephomene requires rapid inoculation of living cultures, and cryopreserved culture strains have not been established in public culture collections. RESULTS: To establish cryopreserved culture strains of two species of Astrephomene, conditions for cryopreservation of the two species were investigated using immature and mature vegetative colonies and two cryoprotectants: N,N-dimethylformamide (DMF) and hydroxyacetone (HA). Rates of cell survival of the A. gubernaculifera or A. perforata strain after two-step cooling and freezing in liquid nitrogen were compared between different concentrations (3 and 6%) of DMF and HA and two types of colonies: immature colonies (small colonies newly released from the parent) and mature colonies (large colonies just before daughter colony formation). The highest rate of survival [11 ± 13% (0.36-33%) by the most probable number (MPN) method] of A. gubernaculifera strain NIES-4017 (established in 2014) was obtained when culture samples of immature colonies were subjected to cryogenic treatment with 6% DMF. In contrast, culture samples of mature colonies subjected to 3% HA cryogenic treatment showed the highest "MPN survival" [5.5 ± 5.9% (0.12-12%)] in A. perforata. Using the optimized cryopreservation conditions for each species, survival after freezing in liquid nitrogen was examined for six other strains of A. gubernaculifera (established from 1962 to 1981) and another A. perforata strain maintained in the Microbial Culture Collection at the National Institute for Environmental Studies (MCC-NIES). We obtained ≥0.1% MPN survival of the A. perforata strain. However, only two of the six strains of A. gubernaculifera showed ≥0.1% MPN survival. By using the optimal cryopreserved conditions obtained for each species, five cryopreserved strains of two species of Astrephomene were established and deposited in the MCC-NIES. CONCLUSIONS: The optimal cryopreservation conditions differed between the two species of Astrephomene. Cryopreservation of long-term-maintained strains of A. gubernaculifera may be difficult; further studies of cryopreservation of these strains are needed.

Rapid transcriptomic and physiological changes in the freshwater pennate diatom Mayamaea pseudoterrestris in response to copper exposure.

Diatoms function as major primary producers, accumulating large amounts of biomass in most aquatic environments. Given their rapid responses to changes in environmental conditions, diatoms are used for the biological monitoring of water quality and for performing ecotoxicological tests in aquatic ecosystems. However, the molecular basis for their toxicity to chemical compounds remains largely unknown. Here, we sequenced the genome of a freshwater diatom, Mayamaea pseudoterrestris NIES-4280, which has been proposed as an alternative strain of Navicula pelliculosa UTEX 664 for performing the Organisation for Economic Co-operation and Development ecotoxicological test. This study shows that M. pseudoterrestris has a small genome and carries the lowest number of genes among freshwater diatoms. The gene content of M. pseudoterrestris is similar to that of the model marine diatom, Phaeodactylum tricornutum. Genes related to cell motility, polysaccharide metabolism, oxidative stress alleviation, intracellular calcium signalling, and reactive compound detoxification showed rapid changes in their expression patterns in response to copper exposure. Active gliding motility was observed in response to copper addition, and copper exposure decreased intracellular calcium concentration. These findings enhance our understanding of the environmental adaptation of diatoms, and elucidate the molecular basis of toxicity of chemical compounds in algae.

Cryopreservation of vegetative cells and zygotes of the multicellular volvocine green alga Gonium pectorale.

BACKGROUND: Colonial and multicellular volvocine green algae have been extensively studied recently in various fields of the biological sciences. However, only one species (Pandorina morum) has been cryopreserved in public culture collections. RESULTS: Here, we investigated conditions for cryopreservation of the multicellular volvocine alga Gonium pectorale using vegetative colonies or cells and zygotes. Rates of vegetative cell survival in a G. pectorale strain after two-step cooling and freezing in liquid nitrogen were compared between different concentrations (3% and 6%) of the cryoprotectant N,N-dimethylformamide (DMF) and two types of tubes (0.2-mL polymerase chain reaction tubes and 2-mL cryotubes) used for cryopreservation. Among the four conditions investigated, the highest rate of survival [2.7 ± 3.6% (0.54-10%) by the most probable number (MPN) method] was obtained when 2.0-mL cryotubes containing 1.0 mL of culture samples with 6% DMF were subjected to cryogenic treatment. Using these optimized cryopreservation conditions, survival rates after freezing in liquid nitrogen were examined for twelve other strains of G. pectorale and twelve strains of five other Gonium species. We obtained ≥ 0.1% MPN survival in nine of the twelve G. pectorale strains tested. However, < 0.1% MPN survival was detected in eleven of twelve strains of five other Gonium species. In total, ten cryopreserved strains of G. pectorale were newly established in the Microbial Culture Collection at the National Institute for Environmental Studies. Although the cryopreservation of zygotes of volvocine algae has not been previously reported, high rates (approximately 60%) of G. pectorale zygote germination were observed after thawing zygotes that had been cryopreserved with 5% or 10% methanol as the cryoprotectant during two-step cooling and freezing in liquid nitrogen. CONCLUSIONS: The present study demonstrated that cryopreservation of G. pectorale is possible with 6% DMF as a cryoprotectant and 1.0-mL culture samples in 2.0-mL cryotubes subjected to two-step cooling in a programmable freezer.

What Happened before Losses of Photosynthesis in Cryptophyte Algae?

In many lineages of algae and land plants, photosynthesis was lost multiple times independently. Comparative analyses of photosynthetic and secondary nonphotosynthetic relatives have revealed the essential functions of plastids, beyond photosynthesis. However, evolutionary triggers and processes that drive the loss of photosynthesis remain unknown. Cryptophytes are microalgae with complex plastids derived from a red alga. They include several secondary nonphotosynthetic species with closely related photosynthetic taxa. In this study, we found that a cryptophyte, Cryptomonas borealis, is in a stage just prior to the loss of photosynthesis. Cryptomonas borealis was mixotrophic, possessed photosynthetic activity, and grew independent of light. The plastid genome of C. borealis had distinct features, including increases of group II introns with mobility, frequent genome rearrangements, incomplete loss of inverted repeats, and abundant small/medium/large-sized structural variants. These features provide insight into the evolutionary process leading to the loss of photosynthesis.

Genome sequencing of the multicellular alga Astrephomene provides insights into convergent evolution of germ-soma differentiation.

Germ-soma differentiation evolved independently in many eukaryotic lineages and contributed to complex multicellular organizations. However, the molecular genetic bases of such convergent evolution remain unresolved. Two multicellular volvocine green algae, Volvox and Astrephomene, exhibit convergent evolution of germ-soma differentiation. The complete genome sequence is now available for Volvox, while genome information is scarce for Astrephomene. Here, we generated the de novo whole genome sequence of Astrephomene gubernaculifera and conducted RNA-seq analysis of isolated somatic and reproductive cells. In Volvox, tandem duplication and neofunctionalization of the ancestral transcription factor gene (RLS1/rlsD) might have led to the evolution of regA, the master regulator for Volvox germ-soma differentiation. However, our genome data demonstrated that Astrephomene has not undergone tandem duplication of the RLS1/rlsD homolog or acquisition of a regA-like gene. Our RNA-seq analysis revealed the downregulation of photosynthetic and anabolic gene expression in Astrephomene somatic cells, as in Volvox. Among genes with high expression in somatic cells of Astrephomene, we identified three genes encoding putative transcription factors, which may regulate somatic cell differentiation. Thus, the convergent evolution of germ-soma differentiation in the volvocine algae may have occurred by the acquisition of different regulatory circuits that generate a similar division of labor.

Genome sequencing of the NIES Cyanobacteria collection with a focus on the heterocyst-forming clade.

Cyanobacteria are a diverse group of Gram-negative prokaryotes that perform oxygenic photosynthesis. Cyanobacteria have been used for research on photosynthesis and have attracted attention as a platform for biomaterial/biofuel production. Cyanobacteria are also present in almost all habitats on Earth and have extensive impacts on global ecosystems. Given their biological, economical, and ecological importance, the number of high-quality genome sequences for Cyanobacteria strains is limited. Here, we performed genome sequencing of Cyanobacteria strains in the National Institute for Environmental Studies microbial culture collection in Japan. We sequenced 28 strains that can form a heterocyst, a morphologically distinct cell that is specialized for fixing nitrogen, and 3 non-heterocystous strains. Using Illumina sequencing of paired-end and mate-pair libraries with in silico finishing, we constructed highly contiguous assemblies. We determined the phylogenetic relationship of the sequenced genome assemblies and found potential difficulties in the classification of certain heterocystous clades based on morphological observation. We also revealed a bias on the sequenced strains by the phylogenetic analysis of the 16S rRNA gene including unsequenced strains. Genome sequencing of Cyanobacteria strains deposited in worldwide culture collections will contribute to understanding the enormous genetic and phenotypic diversity within the phylum Cyanobacteria.

Development of a time-series shotgun metagenomics database for monitoring microbial communities at the Pacific coast of Japan.

Although numerous metagenome, amplicon sequencing-based studies have been conducted to date to characterize marine microbial communities, relatively few have employed full metagenome shotgun sequencing to obtain a broader picture of the functional features of these marine microbial communities. Moreover, most of these studies only performed sporadic sampling, which is insufficient to understand an ecosystem comprehensively. In this study, we regularly conducted seawater sampling along the northeastern Pacific coast of Japan between March 2012 and May 2016. We collected 213 seawater samples and prepared size-based fractions to generate 454 subsets of samples for shotgun metagenome sequencing and analysis. We also determined the sequences of 16S rRNA (n = 111) and 18S rRNA (n = 47) gene amplicons from smaller sample subsets. We thereafter developed the Ocean Monitoring Database for time-series metagenomic data ( http://marine-meta.healthscience.sci.waseda.ac.jp/omd/ ), which provides a three-dimensional bird's-eye view of the data. This database includes results of digital DNA chip analysis, a novel method for estimating ocean characteristics such as water temperature from metagenomic data. Furthermore, we developed a novel classification method that includes more information about viruses than that acquired using BLAST. We further report the discovery of a large number of previously overlooked (TAG)n repeat sequences in the genomes of marine microbes. We predict that the availability of this time-series database will lead to major discoveries in marine microbiome research.

Identification of a dual orange/far-red and blue light photoreceptor from an oceanic green picoplankton.

Photoreceptors are conserved in green algae to land plants and regulate various developmental stages. In the ocean, blue light penetrates deeper than red light, and blue-light sensing is key to adapting to marine environments. Here, a search for blue-light photoreceptors in the marine metagenome uncover a chimeric gene composed of a phytochrome and a cryptochrome (Dualchrome1, DUC1) in a prasinophyte, Pycnococcus provasolii. DUC1 detects light within the orange/far-red and blue spectra, and acts as a dual photoreceptor. Analyses of its genome reveal the possible mechanisms of light adaptation. Genes for the light-harvesting complex (LHC) are duplicated and transcriptionally regulated under monochromatic orange/blue light, suggesting P. provasolii has acquired environmental adaptability to a wide range of light spectra and intensities.

The Rubisco small subunits in the green algal genus Chloromonas provide insights into evolutionary loss of the eukaryotic carbon-concentrating organelle, the pyrenoid.

BACKGROUND: Pyrenoids are protein microcompartments composed mainly of Rubisco that are localized in the chloroplasts of many photosynthetic organisms. Pyrenoids contribute to the CO2-concentrating mechanism. This organelle has been lost many times during algal/plant evolution, including with the origin of land plants. The molecular basis of the evolutionary loss of pyrenoids is a major topic in evolutionary biology. Recently, it was hypothesized that pyrenoid formation is controlled by the hydrophobicity of the two helices on the surface of the Rubisco small subunit (RBCS), but the relationship between hydrophobicity and pyrenoid loss during the evolution of closely related algal/plant lineages has not been examined. Here, we focused on, the Reticulata group of the unicellular green algal genus Chloromonas, within which pyrenoids are present in some species, although they are absent in the closely related species. RESULTS: Based on de novo transcriptome analysis and Sanger sequencing of cloned reverse transcription-polymerase chain reaction products, rbcS sequences were determined from 11 strains of two pyrenoid-lacking and three pyrenoid-containing species of the Reticulata group. We found that the hydrophobicity of the RBCS helices was roughly correlated with the presence or absence of pyrenoids within the Reticulata group and that a decrease in the hydrophobicity of the RBCS helices may have primarily caused pyrenoid loss during the evolution of this group. CONCLUSIONS: Although we suggest that the observed correlation may only exist for the Reticulata group, this is still an interesting study that provides novel insight into a potential mechanism determining initial evolutionary steps of gain and loss of the pyrenoid.

Comparative genome analysis of test algal strain NIVA-CHL1 (Raphidocelis subcapitata) maintained in microalgal culture collections worldwide.

Raphidocelis subcapitata is one of the most frequently used species for algal growth inhibition tests. Accordingly, many microalgal culture collections worldwide maintain R. subcapitata for distribution to users. All R. subcapitata strains maintained in these collections are derived from the same cultured strain, NIVA-CHL1. However, considering that 61 years have passed since this strain was isolated, we suspected that NIVA-CHL1 in culture collections might have acquired various mutations. In this study, we compared the genome sequences among NIVA-CHL1 from 8 microalgal culture collections and one laboratory in Japan to evaluate the presence of mutations. We found single-nucleotide polymorphisms or indels at 19,576 to 28,212 sites per strain in comparison with the genome sequence of R. subcapitata NIES-35, maintained at the National Institute for Environmental Studies, Tsukuba, Japan. These mutations were detected not only in non-coding but also in coding regions; some of the latter mutations may affect protein function. In growth inhibition test with 3,5-dichlorophenol, EC50 values varied 2.6-fold among the 9 strains. In the ATCC 22662-2 and CCAP 278/4 strains, we also detected a mutation in the gene encoding small-conductance mechanosensitive ion channel, which may lead to protein truncation and loss of function. Growth inhibition test with sodium chloride suggested that osmotic regulation has changed in ATCC 22662-2 and CCAP 278/4 in comparison with NIES-35.

Draft Genome Sequences of Four Microcystis aeruginosa Strains (NIES-3787, NIES-3804, NIES-3806, and NIES-3807) Isolated from Lake Kasumigaura, Japan.

Microcystis aeruginosa is a bloom-forming cyanobacterium found in freshwater environments. The draft genomes of the M. aeruginosa strains NIES-3787, NIES-3804, NIES-3806, and NIES-3807, which were isolated from Lake Kasumigaura, Japan, were sequenced. The genome sizes of NIES-3787, NIES-3804, NIES-3806, and NIES-3807 were 4,524,637, 4,522,701, 4,370,004, and 4,378,226 bp, respectively.

Genomic Characteristics of the Toxic Bloom-Forming Cyanobacterium Microcystis aeruginosa NIES-102.

Microcystis aeruginosa, a bloom-forming cyanobacterium distributed mainly in freshwater environments, can be divided into at least 12 groups (A-K and X) based on multi-locus phylogenetic analyses. In this study, we characterized the genome of microcystin-producing M. aeruginosa NIES-102, assigned to group A, isolated from Lake Kasumigaura, Japan. The complete genome sequence of M. aeruginosa NIES-102 comprised a 5.87-Mbp circular chromosome containing 5,330 coding sequences. The genome was the largest among all sequenced genomes for the species. In a comparison with the genome of M. aeruginosa NIES-843, which belongs to the same group, the microcystin biosynthetic gene cluster and CRISPR-Cas locus were highly similar. A synteny analysis revealed small-scale rearrangements between the two genomes. Genes encoding transposases were more abundant in these two genomes than in other Microcystis genomes. Our results improve our understanding of structural genomic changes and adaptation to a changing environment in the species.

Draft Genome Sequences of Three Filamentous Cyanobacterial Strains, Dolichospermum planctonicum NIES-80, Planktothrix agardhii NIES-905, and Sphaerospermopsis reniformis NIES-1949.

Three freshwater planktonic filamentous cyanobacterial strains, Dolichospermum planctonicum NIES-80, Planktothrix agardhii NIES-905, and Sphaerospermopsis reniformis NIES-1949, were sequenced. The genome sizes of NIES-80, NIES-905, and NIES-1949 were 4,571,002 bp, 5,512,454 bp, and 6,025,023 bp, and the number of protein-coding genes in each genome was 4,009, 4,925, and 5,408, respectively.

Depth-dependent transcriptomic response of diatoms during spring bloom in the western subarctic Pacific Ocean.

Diatoms play important roles in primary production and carbon transportation in various environments. Large-scale diatom bloom occurs worldwide; however, metabolic responses of diatoms to environmental conditions have been little studied. Here, we targeted the Oyashio region of the western subarctic Pacific where diatoms bloom every spring and investigated metabolic response of major diatoms to bloom formation by comparing metatranscriptomes between two depths corresponding to different bloom phases. Thalassiosira nordenskioeldii and Chaetoceros debilis are two commonly occurring species at the study site. The gene expression profile was drastically different between the surface (late decline phase of the bloom; 10 m depth) and the subsurface chlorophyll maximum (SCM, initial decline phase of the bloom; 30 m depth); in particular, both species had high expression of genes for nitrate uptake at the surface, but for ammonia uptake at the SCM. Our culture experiments using T. nordenskioeldii imitating the environmental conditions showed that gene expression for nitrate and ammonia transporters was induced by nitrate addition and active cell division, respectively. These results indicate that the requirement for different nitrogen compounds is a major determinant of diatom species responses during bloom maturing.